The nature of neuronal signaling is substantially similar in all animals, ranging from simple invertebrates to man. Neuronal signals take the form of electrical impulses, generated by a change in electrical potential across the plasma membrane and propagated along the characteristically extended neuron. Individual neurons, however, are separated by gaps known as synapses which present a barrier to neuronal signaling in its electrical form. At the synapse, the signal takes the form of a chemical message relayed by a group of small signaling molecules known as neurotransmitters. Many different types of neurotransmitters have been identified, including dopamine (DA), norepinephrine (NE), xcex3-aminobutyric acid (GABA) and serotonin (5HT).
Neurotransmitters are stored in synaptic vesicles located in the presynaptic terminals of nerve cells. Activation of a neuron results in the generation of an electrical signal which travels the length of the cell as an action potential until it reaches the presynaptic terminal. The change in membrane potential in the terminal causes synaptic vesicles to fuse with the nerve cell membrane, prompting the release of neurotransmitter into the synaptic cleft. After traversing the cleft by diffusion, neurotransmitter binds to highly selective receptors on the membrane of the postsynaptic neuron. The nature of the postsynaptic response is dictated by the particular neurotransmitter. Excitatory neurotransmitters cause depolarization of the postsynaptic cell membrane, triggering an action potential that recreates the signal electrically in the postsynaptic neuron. In contrast, inhibitory neurotransmitters suppress activation of the postsynaptic neuron by inhibiting the formation of an action potential.
The extent of the signaling response is controlled by both the quantity of neurotransmitter released and the duration of its activity in the synapse. Many mechanisms ensure removal of the neurotransmitter from the synaptic cleft, including enzymatic destruction, active transport or reabsorption into the presynaptic neuron. Active transport is mediated by a class of transporter proteins, each specific to a particular type of neurotransmitter.
Drugs that alter brain function may impact synaptic signaling. Drugs such as cocaine and Prozac(trademark), for example, alter brain activity by blocking the normal function of transporter proteins. Cocaine is a nonselective amine transporter antagonist which exerts its primary addictive effect by blocking dopamine transporter function. Prozac(trademark) belongs to a class of compounds known as serotonin reuptake inhibitors, or SRRIs, that enhance the mood-elevating effects of 5HT by preventing its reabsorption into the presynaptic neuron. Other mood-altering drugs target different neurotransmitters, including Edronax(trademark), which is used to treat severe depression and functions by altering NE levels.
A family of proteins specialized for transport and reputake of neurotransmitters has been identified and cloned. Known as the GABA/norepinephrine transporter (GAT1/NET) gene family, this group of proteins is characterized by identity of amino acid sequence as well as similarity of predicted topographies. Specifically, transporters contain 12 hydrophobic regions thought to form transmembrane domains.
Recently, multiple genes were identified in the nematode C. elegans as having significant homology to the GAT1/NET gene family. See Wilson et. al., Nature 368, 32 (1994). One such gene, T23G5.5, is located on chromosome 3, spanning cosmids CET23G5 and CET02C1. The inferred translation of T23G5.5 has high sequence similarity to a subgroup of the GAT1/NET family, the biogenic amine neurotransmitter transporters. This subgroup includes dopamine transporters (DATs), norepinephrine transporters (NE) and serotonin transporters (SERTs).
The profound behavioral effects of exogenous dopamine in C. elegans, including inhibition of locomotion [Schafer, W. R. et al. Genetics 143, 1219 (1996)] and egg laying [Horvitz, H. R. et al., Science 216, 1012 (1982)] indicate that dopamine uptake blockers are suitable as antihelmintic agents. Broader pesticidal effects of monarnine uptake blockers are suggested by flies with mutations in the gene inebriated, which encodes a homologue of the GAT1/NET family of transporters. These mutants display altered motor coordination in response to anesthetics.
Dopamine uptake blockers identified in high-throughput screens using heterologously expressed cDNAs of the present invention are suitable anti-parasitic agents. The cDNAs themselves are useful for synthesizing suitable probes in molecular diagnostics. The proteins expressed by the cDNAs of the present invention, CeDAT1 and CeDAT2, are useful antigens for generating specific antibodies.
It is therefore an object of the present invention to provide a nucleic acid sequence encoding a functional catecholamine/dopamine transporter from C. elegans. 
It is a further object of the present invention to provide a nucleic acid sequence to make probes for the same protein in other species and related proteins.
It is a further object of the present invention to provide a protein, as well as an antibody, useful in research and molecular diagnostics.
It is a still further object of the present invention to provide a protein useful in the design of therapeutic transporter modulators for clinical treatment.
It is yet a further object of the present invention to provide a protein useful in the identification or design of antihelmintic compounds, as well as new psychoactive drugs.
These and other objects and advantages are obtained by the present invention, which relates to cDNA from the nematode C. elegans encoding at least two proteins, CeDAT1 and CeDAT2, with significant homology to mammalian biogenic amine neurotransmitter transport proteins is described. The sequences were not predictable from known DNA data bank sequences. CeDAT1 is a shortened form of CeDAT2, with 19 amino acids removed from the NH2 terminus of CeDAT2. The sequence of the cDNA has been employed to construct transfected cellular expression systems, which are useful as screening assays for psychoactive drugs that interact with mammalian biogenic amine neurotransmitter transport proteins.